Correction of the Enzyme Deficiency in Hematopoietic Cells of Gaucher Patients Using a Clinically Acceptable Retroviral Supernatant Transduction Protocol
Abstract
Gaucher disease is a lysosomal storage disorder caused by a deficiency of the enzyme glucocerebrosidase (GC), and is an excellent candidate for gene replacement therapy. To develop a clinically acceptable protocol for this purpose, we created two amplified (A) high-titer retroviral vector-producer cell lines to efficiently transduce hematopoietic stem and progenitor cells. GP+envAml2/A-LGSN (A-LGSN), contained the GC cDNA driven by the retroviral long terminal repeat (LTR) and the neomycin phosphotransferase gene expressed from the simian virus 40 early promoter. GP+envAml2/ALG4 (A-LG4) contained only the GC gene driven by the LTR. Both A-LGSN and A-LG4 contained multiple-proviral copies and gave approximately 10-fold higher titers on 3T3 cells compared to their unamplified counterparts. These vectors were packaged in GP+envAml2 cells because vectors produced in this cell line transduced hematopoietic cells more efficiently than other packaging cells tested. Bone marrow mononuclear cells and purified CD34+ cells were infected with virus supernatants four times in the presence of interleukin-3 (IL-3), IL-6, and stem cell factor (SCF) over 96 hours in culture. Cells %%,ere then plated in semisolid cultures and colony-forming unit-granulocyte/macrophage (CFU-GM) colonies were scored for vector presence by polymerase chain reaction (PCR). Transduction efficiency of CFU-GM colonies derived from CD34- cells, as improved considerably using the amplified vectors in the GP+envAm12 packaging line. For A-LGSN, A-LG4, and unamplified LGSN, transduction efficiencies were 41, 42, and 25%, respectively. Gene therapy, CD34+, Stem cells, CC Deficiency, Gaucher disease, Gene therapy, Retroviral supernatant, Transduction
Document Details
- Document Type
- Technical Report
- Publication Date
- Jan 01, 1994
- Accession Number
- ADA279196
Entities
People
- Harish P. Dave
- Licheng Xu
- Pamela H. Correll
- Raphael Schiffmann
- Stefan Karlsson
- Stephanie K. Stahl
- Steven Kessler
Organizations
- Naval Medical Research Center